The long-term goal of this project is to determine the cellular mechanisms involved in the detection and discrimination of taste stimuli by receptor cells. Using various configurations of the patch-clamp recording technique and electron microscopy combined with electrophysiological recording, the following specific aims will be addressed: 1. TO CHARACTERIZE THE EFFECT OF DIFFERENT TASTE STIMULI ON WHOLE-CELL CONDUCTANCES. The cellular mechanisms used by taste cells to detect bitter compounds, amino acids, glutathione and various salts will be determined by using giga-seal whole-cell recording. For each tastant it will be determined if ionic conductance changes are involved, if taste stimuli activate or modulate the conductance and if the effects of taste stimuli are direct, or involve G proteins and 2nd messengers. 2. TO DETERMINE THE SUBCELLULAR LOCALIZATION OF RECEPTORS AND CHANNELS INVOLVED IN TASTE TRANSDUCTION. Diverse types of proteins can serve receptive functions in taste cells, such as the voltage-sensitive K+ channels which we have shown to be restricted to the apical membrane. Are other types of taste "receptors" similarly restricted to the apical membrane of the taste cells? Loose-patch and whole-cell recording techniques will be used to map the distribution of (1) ligand-gated channels with receptors for taste stimuli, (2) taste receptors coupled to second messenger transduction mechanisms and (3) other channels involved in taste transduction. 3. TO CHARACTERIZE THE EFFECT OF DIFFERENT TASTE STIMULI ON SINGLE CHANNELS. The cellular mechanisms used by taste cells to detect taste stimuli will be characterized further using single-channel recording techniques to determine (1) the specific ion channels that are activated or modulated during taste transduction and (2) the role of G proteins and 2nd messengers. Together with Specific Aim #1, these experiments will establish the mechanism of transduction for different taste modalities.l 4. TO DETERMINE IF TASTE DISCRIMINATION OCCURS AT THE LEVEL OF THE RECEPTOR CELL. Taste cells will be studied with electron microscopy following whole-cell recording to determine (1) if the differences in membrane properties observed in taste cells can be attributed to morphologically-identifiable criteria, such as taste cell type, and (2) if specific cell types respond differentially to taste stimuli. Taken together, these studies will establish the whole-cell response to different categories of taste stimuli, the mechanisms of these responses, and the identity and localization of the receptors and channels involved. Additionally, these studies will determine whether taste discrimination involves distinct types of taste cells responding differentially to taste stimuli. Such information will not only enhance understanding of chemosensory transduction, but also provide clues to basic mechanisms involved in signal transduction.